Abstract

The mechanisms underlying neoplastic epithelial cell killing by ionizing
radiation are largely unknown. We discovered a novel response to
radiation manifested by autophagy and the development of acidic
vesicular organelles (AVO). Acidification of AVO was mediated by the
vacuolar H+-ATPase. Staining with the lysosomotropic
agent acridine orange enabled us to quantify AVO accumulation and to
demonstrate their time- and dose-dependent appearance. The appearance
of AVO occurred in the presence of the pan-caspase inhibitor
z-Val-Ala-Asp(Ome)-fluoromethyl ketone, but was inhibited by
3-methyladenine, an inhibitor of autophagy. The accretion of AVO in
surviving progenies of irradiated cells, and the increased incidence of
clonogenic death after inhibition of vacuolar H+-ATPase
suggest that formation of acidic organelles represents a novel defense
mechanism against radiation damage.

Introduction

The cellular and molecular processes involved in the response of
neoplastic epithelial cells to radiation are largely unknown. Whereas
in some cell types, particularly cells of reticuloendothelial origin,
death after irradiation is preceded by apoptotic changes, apoptosis
plays little or no role in the killing of epithelial neoplastic cells
by radiation
(1,
2,
3)
. Epithelial cells do not
undergo apoptosis after irradiation and are likely to respond with a
different sequence of programmed cytoplasmic and nuclear events.
Several investigators have proposed two types of programmed cell death
(4, 5)
. Type I programmed cell death, or apoptosis, is
mediated by a cascade of cysteine aspartases (caspases) and factors
released by the mitochondria
(6)
, and it has typical
morphological and biochemical characteristics such as chromatin
margination and condensation, early nuclear collapse, and nucleosomal
ladder formation
(5)
. In contrast, type II programmed cell
death is marked morphologically by increased autophagy and early
destruction of the cytoplasm that occurs either without nuclear
collapse or precedes it
(5)
. Type II programmed cell death
has been documented mainly in the Lepidoptera during
metamorphosis and during involution of the rat mammary gland
(4, 5)
, but it has rarely been associated with stress-inducing
stimuli
(7, 8)
. Unfortunately, methods for
quantification of type II programmed cell death are lacking, and the
molecular mechanisms that regulate it have not been defined. The work
presented here characterizes and quantifies a novel form of response to
radiation in cancer cells that is reminiscent of type II programmed
cell death. This response is dominated by the appearance and
accumulation of
AVO.
3
Interference with the acidification of these AVO results in
increased radiosensitivity and thus identifies a new target for
modulating the radiation response of cancer cells.

Materials and Methods

Cell Culture.

MCF-7 (human breast adenocarcinoma), LoVo (human colon adenocarcinoma),
and LNCaP (human prostate carcinoma) were obtained from the American
Type Culture Collection. Cells were maintained as described previously
(9)
. Irradiation was carried out 48 h post-plating
(time 0) at 25°C using a Cs-137 irradiator (Shepherd Mark-I,
model 68, SN643) at a dose-rate of 243 cGy/min. Bovine aortic
endothelial cells were obtained from Dr. Haimovitz-Friedman,
Memorial Sloan Kettering Cancer Center, New York) and were grown and
treated with H2O2 as
described previously
(10, 11)
. PMA (Alexis Biochemicals
Corporation, San Diego, CA) was dissolved in DMSO and added to cells
for the duration of 1 h. Final DMSO concentration was 0.03%. The
cells were then rinsed with warm growth medium before being returned to
the incubator for an additional 48 h.

Supravital Cell-staining with Acridine Orange.

Cell staining was performed according to published procedures
(12,
13,
14)
. Acridine orange (Polysciences, Warrington, PA)
was added at a final concentration of 1 μg/ml for a period of 15
min. Bafilomycin A1 (Sigma Chemical Co., St. Louis, MO) was
dissolved in DMSO and added to the cells 30 min before addition of
acridine orange. LysoSensor Blue DND-167 (Molecular Probes, Eugene, OR)
was added for 8 min at a final concentration of 10μ
m. Pictures were obtained with a fluorescence
microscope (Olympus BH-2 RFCA) equipped with a mercury 100-W
lamp, 490-nm band-pass blue excitation filters, a 500-nm dichroic
mirror, and a 515-nm-long pass-barrier filter. Images of control and
irradiated cells were recorded on Kodak Elite II 100 ASA film for color
slides by 4-s exposure.

In acridine orange-stained cells, the cytoplasm and nucleolus fluoresce
bright green and dim red, whereas acidic compartments fluoresce bright
red
(13, 14)
. The intensity of the red fluorescence is
proportional to the degree of acidity and/or the volume of the cellular
acidic compartment
(14)
. Therefore, by comparing the mean
red:green fluorescence ratio within different cell populations, we
could measure a change in the degree of acidity and/or the fractional
volume of their cellular acidic compartment. Cells were stained with
acridine orange for 17 min, removed from the plate with trypsin-EDTA,
and collected in phenol red-free growth medium. Green (510–530 nm) and
red (>650 nm) fluorescence emission from 104
cells illuminated with blue (488 nm) excitation light was measured with
a FACSCalibur from Becton Dickinson (San Jose, CA) using CellQuest
software. The red:green fluorescence ratio for individual cells was
calculated using FlowJo software (TREE STAR, Inc., San Carlos, CA). To
control for the possible effect of trypsinization on the measured
red:green fluorescence ratio, we compared the ratios obtained by flow
cytometry with those obtained with a Laser Scanning Microscope (LSM510;
Zeiss). Stained cells, grown on coverglass, were illuminated with a
488-nm argon laser beam. The red (>650 nm):green (505–545 nm)
fluorescence ratio of an entire image was obtained using software LSM
510 version 2.01 SP2. These measurements yielded similar results to
those obtained with flow cytometry. All determinations of red:green
fluorescence ratio reported here were therefore obtained via flow
cytometry.

Electron Microscopy.

Cell processing for electron microscopy and staining with DAMP
(Molecular Probes, Eugene, OR) was done according to published
procedures
(15, 16)
. The fraction of the cytoplasmic
volume occupied by AVO (the fractional volume of AVO) was quantified
from electron micrographs according to Dunn
(16)
and Lenk
et al.(17)
. Digital images of the micrographs
were obtained with an Epson ES-1200S flat bed scanner with Adobe
Photoshop version 5. The fractional volume was calculated with Image
Pro Plus version 3 and expressed as a percentage of total cytoplasmic
volume.

Detection of Nucleosomal Fragmentation of Genomic DNA.

DNA extraction and electrophoresis on agarose gel was carried out
according to Bose et al.(18)
. DNA preparation
and resolution with pulse field gel electrophoresis was
conducted as described by Gilles et al.(19)
using the CHEF Mapper (Bio-Rad, Richmond, CA). DNA strand breaks were
assayed by the TUNEL method and analyzed by flow cytometry
(10)
.

Surviving Fraction.

Cells were plated in growth medium at a density of 30
cells/cm2 and irradiated 22 h later with 2
and 3 Gy. Cells were irradiated at room temperature in a Cs-137
Irradiator (Sheperd Mark-I, Model 68, SN 643) at a rate of 2.5 Gy/min.
Six days later, 90–95% of the grown colonies possessed >50 cells.
For determination of their red:green ratio, colonies were processed as
described above. For determination of surviving fractions, cells were
stained with crystal violet and colonies containing ≥50 cells
were counted with a dissecting microscope. The surviving fraction was
defined as the ratio between the number of surviving colonies in
irradiated culture and in unirradiated culture, and it was calculated
at each dose level
(21)
.

Results and Discussion

Our experiments indicated that human breast cancer cells are
sensitive to standard doses of radiation. After irradiation with 6 Gy,
only 0.1% of the cells remained clonogenic
(22)
.
Nonetheless, the cells did not show any of the biochemical and
morphological changes that are associated with apoptosis up to 4 days
after irradiation with 10 Gy. At 4 days after irradiation, 30% of the
cells were already dead and did not exclude trypan blue. Still,
nucleosomal ladder formation and a positive TUNEL reaction could not be
demonstrated (Fig. 1)
⇓
. Furthermore, electron microscopy did not reveal the morphological
changes that are typical of apoptosis, i.e., chromatin
margination and condensation (Fig. 2)
⇓
. Instead, DNA damage was manifested by micronuclei formation
(9)
and nondiscrete DNA degradation (Fig. 1)
⇓
. Our findings
and the absence of apoptotic markers after irradiation of malignant
epithelial cells reported by others
(1, 2)
led us to
search for type II programmed cell death-related cellular events, such
as changes in the cellular acidic compartments.

Ultrastructure of AVO formed in irradiated cells.
A, a, control unirradiated cells 48 h postirradiation time (Time 0). b, cells irradiated
with 10 Gy, 48 h postirradiation. The arrow points
to newly formed AVO. Bar
(a–b), 4 μm. B,
a–e, newly formed vesicular organelles
in cells irradiated as above. The arrows point to the
part-rough, part-smooth membrane cisternae (a), to
vesicles fusing with membrane cisternae (b), to lamellar
structures (c and d), and to residual
digested material (e). Bar
(a–e), 0.6 μm. C,
concentration of the lysosomotropic agent DAMP in AVO (AVO-EM)
demonstrated by immunogold histochemistry. Cells were stained with DAMP
24 h postirradiation with 10 Gy and processed for viewing as
described in “Materials and Methods.” The arrow
points to the gold particles over the AVO (a). Cells
were incubated with 0.5 μm bafilomycin A1
(b) or with 300 μm chloroquine
(c) before the addition of damp.
Bar (a–c), 0.6 μm.
D, distribution of unirradiated and irradiated cell
populations according to the fraction of the cytoplasmic volume
occupied by AVO-EM (fractional volume). Fractional volume of AVO-EM was
calculated as described in “Materials and Methods.”

For detecting of the acidic compartment, we used the lysosomotropic
agent acridine orange, a weak base that moves freely across biological
membranes when uncharged. Its protonated form accumulates in acidic
compartments, where it forms aggregates that fluoresce bright red
(12,
13,
14)
. Vital staining of MCF-7 cells with acridine
orange revealed the appearance of AVO after irradiation.
Concentrated dye in the vesicles fluoresced bright red, whereas the
cytoplasm and the nucleus showed dominant green fluorescence (Fig. 3
⇓
Ab). In contrast, the majority of unirradiated cells exhibited mainly
green fluorescence with minimal red fluorescence (Fig. 3Aa⇓
).
In numerous studies, demonstration of vacuolar
H+-ATPase-dependent acidification of cellular
organelles, as well as its involvement in different cellular
processes, was achieved by using its specific inhibitor bafilomycin A1
(23, 24)
. Similarly, by addition of the inhibitor to MCF-7
cells, we were able to demonstrate that acidification of AVO is
mediated by the vacuolar H+-ATPase (Fig. 3A, b and c⇓
; Ref.
23
). Preincubation
of the cells with 300 μm of the weak amine
chloroquine also inhibited acridine orange accumulation in AVO (data
not shown).

To measure the radiation-induced increase in fractional volume and/or
acidity of AVO, we determined the mean red:green fluorescence ratio in
control and irradiated cells. At 24 h after irradiation, 92% of
the unirradiated controls were tightly distributed around their mean
red:green fluorescence ratio (Fig. 3Ba⇓
). Only 8% had a
red:green fluorescence ratio that increased asymptotically above 2 (the
highest value in the descending limb of the histogram). On the other
hand, 55% of the irradiated cells had a red:green ratio that was >2,
and the mean value of red:green fluorescence ratio was 2.3-fold higher
than in controls (Fig. 3Bb⇓
). Bafilomycin A1 decreased the
mean red:green fluorescence ratio in unirradiated cells and also
inhibited its radiation-induced increase. In the presence of
bafilomycin A1, the mean red:green fluorescence ratio was similar in
both unirradiated and irradiated cells, indicating that the
radiation-induced increase in this ratio is attributable to the
development of AVO, rather than other possible changes in the molecular
composition of the irradiated cells (Fig. 3B, c and d)
⇓
. AVO appearance was dependent upon the radiation dose
and increased with the time after irradiation (Fig. 4)
⇓
.

Increased red:green (R/G) fluorescence
ratio in irradiated cells is radiation dose- and time-dependent. Cells
were stained and processed for flow cytometric analysis. The numbers
represent the fold increase of the red:green fluorescence ratio in
irradiated cells above controls, and are the mean ± SD
of triplicate samples from one experiment that was reproduced twice
with similar results.

To differentiate further between the acidic compartments of control and
irradiated cells, we used an additional lysosomotropic agent,
LysoSensor Blue DND-167. The fluorescence of LysoSensor Blue is
pH-dependent and increases as pH decreases. In unirradiated cells,
LysoSensor Blue hardly showed any fluorescence, whereas irradiated
cells fluoresced bright blue (Fig. 3A, e and f)
⇓
, indicating that the pH of the acidic compartments in
irradiated cells is indeed lower than that of unirradiated cells. AVO
appearance was associated with increased levels of the lysosomal
membrane protein LAMP-1, as evident from Western blot analysis and
immunocytochemistry (Fig. 5A and B)
⇓
.

Radiation induces increase in LAMP-1 levels.
A, Western blotting analysis of LAMP-1. Cells were
harvested 48 h after irradiation with 10 Gy. Equal amounts of cell
lysates from control and irradiated cells were analyzed for LAMP-1
content. C, control unirradiated cells.
5, cells irradiated with 5 Gy. 10,
Cells irradiated with 10 Gy. B,
immunolocalization of LAMP-1 in control and irradiated cells. The
staining showed the increased levels of LAMP-1 in the cells at 48 h after irradiation and its localization to vesicular bodies.
a, unirradiated cells. b, cells
irradiated with 10 Gy.

The increase in the red:green fluorescence ratio could be modulated by
PMA. The mean red:green fluorescence ratio increased by a factor of
1.7 ± 0.3 (n = 3) 48 h
after stimulation with 30 nm PMA. This result
suggests that protein kinase C may be involved in the increased
red:green fluorescence ratio after irradiation.

The increase in the mean red:green fluorescence ratio after irradiation
was also observed in two other cancer cell lines. Forty-eight h after
irradiation with 10 Gy, the mean red:green fluorescence ratio increased
in prostate cancer (LNCaP) and in colon adenocarcinoma (LoVo) cells by
1.6 ± 0.1 (n = 3) and
2 ± 0.2 (n = 3) -fold,
respectively. As in MCF-7 cells, the increase in the mean red:green
fluorescence ratio in LoVo and LNCaP cells was associated with the
appearance of red fluorescent AVO.

Parallel investigations with electron microscopy confirmed the
radiation-induced formation of a new acidic compartment (Fig. 2, A and B)
⇓
. These subcellular AVO were
composed of core vesicles with granular, vesicular, or lamellar
content. The core vesicles were often surrounded by and intertwined
with smooth or part smooth/part rough membrane cisternae that were
found to fuse with smooth vesicles of unknown origin (Fig. 2B)
⇓
. The diameter of these organelles ranged from 0.5–2.5μ
m and was comparable with the diameter of the largest red
fluorescent AVO in irradiated cells. Because fluorescent AVO may
consist of a heterogeneous population of AVO, we termed the ones
characterized by electron microscopy “AVO-EM.” AVO-EM were found to
be acidic by virtue of their ability to concentrate the lysosomotropic
agent DAMP (Fig. 2C)
⇓
. By 48 h postirradiation with 10
Gy, the average fractional volume of AVO-EM in the population was
16 ± 0.1% (Fig. 2D)
⇓
, whereas, the average
fractional volume in unirradiated cells was 0.91 ± 0.01%. The emergence of AVO-EM during the first 48 h
postirradiation with 2–10 Gy was dose- and time-dependent (data not
shown).

During autophagy, portions of the cytoplasm and subcellular
organelles are sequestered by the endoplasmic reticulum,
resulting in vesicular bodies that are bound by double-membrane
cisternae
(25)
. The association of the core vesicles with
membrane cisternae in AVO-EM bears morphological similarities to
autophagous bodies. We therefore examined the effect of
3-methyladenine, an inhibitor of autophagy
(25, 26)
, on
AVO formation. 3-Methyladenine at a final concentration of 5
mm decreased the red:green ratio at 48 h postradiation
with 10 Gy from 1.77 ± 0.01 to 1.15 ± 0.01 (n = 3). Electron microscopy analysis
demonstrated a parallel reduction in the fraction of cells containing
AVO-EM from 94% to 22%. The effect of 3-methyladenine on irradiated
cells suggests that the formation of AVO after irradiation may share
similar pathways with processes that regulate autophagy.

It is important to note that in addition to ionizing irradiation, other
death-inducing agents such as tumor necrosis factor and
staurosporin kill MCF-7 without producing typical apoptotic changes
(27)
. It has recently been reported that the lack of
apoptotic response to tumor necrosis factor results from the absence of
caspase-3 in these cells
(27)
. The absence of caspase-3
may well explain the lack of apoptotic response to ionizing
irradiation. Nonetheless, the emergence of AVO in the presence of the
pan-caspase inhibitor z-VAD-fmk at concentrations ranging from 50–154μ
m (Table 1)
⇓
suggests that the programmed events that lead to AVO formation are not
related to apoptosis.

z-VAD-fmk (Enzyme Systems Products, Livermore, CA) was added to cells
1 h before irradiation. Cells were harvested 24 h after
irradiation and processed for determination of their mean red:green
fluorescence ratio. Numbers are means ± SD from
triplicate samples of one experiment that was reproduced twice at 50μ
m and 154 μm.

Cells that survive radiation may continue to divide and form colonies,
although their DNA might have sustained damage
(28)
. We
found that the progenies of irradiated cells contain an increased level
of AVO (Table 2)
⇓
. This led us to postulate that the emergence of acidic compartments
protects the cells against radiation damage. In fact, experiments with
bafilomycin A1 showed that inhibition of vacuolar
H+-ATPase, the enzyme that mediates AVO
acidification, augmented DNA degradation and decreased survival after
irradiation (Fig. 6
⇓
; Table 2
⇓
). Addition of bafilomycin A1 2 days after irradiation with 10
Gy, for a period of 24 h, dramatically increased DNA cleavage into
large fragments (20–1000 kb). Also, addition of bafilomycin A1 for
24 h after irradiation with 2 and 3 Gy reduced the surviving
fraction by 30–40% without significantly affecting the survival of
unirradiated cells.

AVO accumulation in progenies of irradiated cells is necessary for
their survival

Red:green ratio in colonies was determined as described in “Materials
and Methods.” The numbers are mean ± SD from three
separate experiments. Bafilomycin A1 was added to irradiated cells at
the time of irradiation for 24 h. The effect of bafilomycin on the
survival of irradiated cells was significant (P < .05;
Student’s t test). The numbers are mean ± SD from one experiment that was reproduced once with similar results.
Colonies in five plates were counted for each dose.

Increased autophagy, the hallmark of programmed cell death type
II, is thought to lead to cell death via destruction of the cytoplasm.
Still, the lysosomal compartment has been linked to cellular defense
mechanisms such as protection against infectious agents
(29)
. Recently, acidic compartments have been associated
with drug resistance of breast cancer cell lines
(30)
, and
in yeast autophagy is required for cell survival during starvation
(31)
. Similarly, our results suggest that accumulation of
AVO after irradiation is modulated by cellular defense
mechanisms. These AVO may protect the cells by preventing cytoplasmic
acidification, by providing catabolites required for repair processes,
and/or by containing toxic molecules. Our experiments show that
moderate formation of AVO in surviving colonies provides long-term
protection against low-radiation damage. However, continuous accretion
of AVO after high levels of damage may offset their protective effect,
leading to replacement of the normal cytoplasm and possibly to necrosis
and cell death. Therefore, inhibition of AVO formation or function may
serve as a tool to increase cell death after low-radiation damage and
facilitate cell-kill after high-radiation damage. Modulation of AVO
function may prove useful for increasing the therapeutic ratio of
radiation treatment of epithelial cancers.

Acknowledgments

We thank Drs. David Golde, Haya Herscovitz, and Victoria
Iwanij for critical reading of the manuscript; John Takawa from
the Molecular Cytology Core Facility at the Memorial Sloan-Kettering
Cancer Center for his help with the morphometric quantifications; Drs.
Fred Gilles and Andre Goy for their help with the pulse field gel
electrophoresis; and Vincent Yeugelowitz for excellent technical
assistance.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

↵1 Supported by grants from the Dewitt-Wallace
Fund, the Sports Foundation Against Cancer, the Connecticut Sports
Foundation, the Reich-Jossem Fund, and a Radiological Society of North
America grant (to T. H.).